Studies On Physiology, Biochemistry And Molecular Biology On Betalain Synthesis In Amaranthus Tricolor

Betalains, which are natural antioxidants with cancer chemopreventive properties, are indole-derived pigments that substitute for anthocyanins in plants of the order Caryophyllales. Amaranthus species are rich in betalains, which make them a valuable alternative to beets as a source of natural betalains. The betalain biosynthesis pathway has not been fully characterized although some of the main steps, possible side reactions, and key enzymes have been identified. Further research is necessary to completely characterize the biosynthetic and metabolic activities associated with these pigments. In this study, five main A. tricolor betalain biosynthesis genes (including 3 glycosyltransferase genes,1 AmaTyDC gene and 1 AmaDODA gene) were identified based on our transcriptome data and were successfully cloned from leaf-vegetable amaranth by using RT-PCR and RACE technology. Promoter sequence of AmaB5-GT and AmaDOPA5-GT also were cloned by using genome walking. The betalain content differences and these genes’ expression levels in different tissues of adult Amaranthus tricolor, in different varieties and in seedlings treated by different phytohormones and light qualities were compared for the characterization of betalain biosynthesis genes. Moreover, to the better understand the betalain biosynthesis molecular mechanism, the transcriptome differences between red leaf part and green leaf part were compared in combination with some physiological and biochemical indices.1 The analysis and cloning of betalain synthesis genes and promoters in Amaranthus tricolorThe cDNA and DNA sequences of 3 glycosyltranferase genes (AmaDOPA5-GT (GenBank accession number:kp174811,1,700 bp), AmaB6-GT(GenBank accession number:kp174812, 1,674 bp), AmaB6-GT (GenBank accession number:kp174810,1,666 bp)),1 decarboxylase gene AmaTyDC (GenBank accession number:kp165397,1,669 bp) and 1 dioxygenase gene AmaDODA (GenBank accession number:kp165399,1,085 bp) by using RT-PCR and RACE technology. Blast result showed that among these 5 genes only AmaDODA has intrones (2 introns) in its DNA sequence.The promoter sequences of AmaDOPA5-GT and AmaB5-GT were successfully cloned by using genome walking technology. Several light-, hormone-, anaerobic-, drought-, and some other stress-related elements and MYB transcription factor binding site were found in both the two promoters, suggesting that the expression of AmaDOPA5-GT and AmaB’5-GT may be regulated by these factors.2 The correlation analysis of betalain content and betalain biosynthesis related genes expression levels in Amaranthus tricolor2.1 The study of betalain content and betalain biosynthesis related genes’expression in different tissues and different amaranth varietiesThere are obvious differences on betalain content in different amaranthus varieties, most betalain content in whole red type amaranth, least in green amaranth. The result of qRT-PCR showed that the expression levels of AmaB6-GT、AmaB5-GT、AmaDOPA5-GT and AmaTyDC were highest in whole red type amaranth, the expression levels AmaDOPA5-GT and AmaTyDC were accordance with betalain content four amaranth varieties, so it suggested that AmaDOPA5-GT and AmaTyDC played an important role in betalain synthesis. There was less change on AmaDODA expression levels in four amaranth varieties, It is possible that the 4,5-DOPA dioxygenase extradiol encoded by AmaDODA is involved in other reactions in addition to those related to betalain synthesis.Different tissues of adult A. tricolor plants accumulated pigments to different levels, with the greatest abundance in red tissues (i.e., red leaf parts and red stems), followed by green-leaf part, red stem, root, green stem, and petiole. However, pigment accumulation did not correlate with gene expression levels in various tissues. The highest expression levels for AmaB5-GT, AmaB6-GT, and AmaDOPA5-GT occurred in the petioles, leaves, and roots/green leaves, respectively.2.2 Effect of different hormones on betalain content and gene expression levels in Amaranthus tricolor seedlingsExogenous cytokinin(KT and 6-BA) can promote betalain content in Amaranthus tricolor seedlings. The genes encoding glycosyl transferases were induced by KT, and the betalain contents also increased in KT-treated plants. In contrast, AmaTyDC transcript levels decreased in KT-treated plants, which resulted in decreased production of the pigment associated with AmaTyDC. The AmaDODA expression levels also decreased following KT treatments. It is possible that AmaDODA is involved in other reactions in addition to those related to betalain synthesis. The transcript levels of AmaDODA, AmaDOPA5-GT, and AmaB5-GT increased with increasing 6-BA concentrations. This result suggested that 6-BA increases DODA activity, thereby increasing the amount of substrate available to form stable amaranthin via the glycosylation activities of AmaDOPA5-GT and AmaB5-GT. However,6-BA did not affect the transcript levels of AmaTyDC and AmaB6-GT.Exogenous applications of 2,4-D and GA3 inhibited betacyanin accumulation in A. tricolor seedlings, The changes in the expression levels of AmaB5-GT, AmaTyDC, and AmaB6-GT were consistent with the decrease in betacyanin contents, the results indicated that the genes induced by exogenous 2,4-D. Exposure to GA3 redirects betalain precursors to other metabolic pathways,This may explain why AmaTyDC expression levels were relatively unchanged following GA3 treatment. The high expression levels of the other four genes were insufficient to increase betacyanin accumulation in A. tricolor seedlings treated with GA3.2.3 Effect of different light qualities on betalain content and gene expression levels in Amaranthus tricolor seedlingsThe betalain content and genes expression levels were studied in A. tricolor seedlings treated different qualities. The result showed that short wavelength light (blue light) promoted betalain accumulation. The transcript levels of AmaTyDC、AmaB5-GT and AmaDOPA5-GT increased in A. tricolor seedlings treated short wavelength light, it indicated that these genes affected betalain biosynthesis. Surprisingly, there were high transcript levels of AmaTyDC, AmaDODA, AmaDOPA5-GT, and AmaB5-GT in the red light treatment, it is possible that feedback regulation was resulted from the decreasing of betalain.The transcript levels of all five genes were high or highest in A. tricolor seedlings treated with blue light, followed by red light, compound light, and white light. The lowest gene transcript levels were in plants in the dark treatment.3 The study of physiology and biochemistry of different tissues and varieties with different betalain content3.1 The study of betalain distributionThe betalain distribution in different tissues of two different varieties (’da hong’ and ’bai yuan ye’ amaranth) was studied by using hand-sliced method. Results showed that betalain accumulated principally in epidermis cell of vacuum side of blade around leaf vein and sheath cell around vascular bundle of different tissues. No betalain was observed in ’bai yuan ye’ amaranth. Interestingly, more starch grains were found in ’bai yuan ye’ than that in ’da hong’ amaranth. And the starch grains were mainly distributed in the part beside leaf vein of ’da hong’ amaranth.3.2 Differences of photosynthetic rate, antioxidant ability, chlorophyll fluorescence between red part leaf and green part leafIn the red part leaf, lower net photosynthetic rate was detected. Higer SOD enzymatic activity and total antioxidant capacity were found in red pat leaf than that in green part leaf, which confirmed the antioxidant ability of betalain.The chlorophyll fluorescence differences of red-leaf part and green-leaf part of’da hong’ amaranth under 25℃ and 40℃ were compared. Results showed that under room temperature (25℃), the photosynthetic capacity of green part leaf was higher than that of red part leaf. Under high temperature treatment (40℃), the photosynthetic capacity of green part leaf reduced more significantly than that of red part leaf, which suggest that betalain has protective effect for amaranth plant.4 Comparative transcriptome analysis of red part leaf and green part leafRNA Seq was applied to analyze the tanscriptome differences between red part leaf and green part leaf.In total, we obtained 80,026 unigenes, among which 1320 were up-regulated and 858 were down-regulated in red part leaf. Gene ontology analysis showed that these genes were mainly involved in secondary metabolism, phytohormone signal transduction, photosynthesis, starch and sucrose metabolism pathway. Results generated in the 2-4 part of this abstract were mostly consistant with our transcriptome data.In red part leaf, flavonoids metabolism related genes were mostly down-regulated. The down-regulation of these genes can partialy explain why there is no anthocyanin exit in betalain rich plans. Many MYB transcription factor family genes showed significant differential expression between the two parts of leaves. Three of these MYB genes showed very high homology with the betalain biosynthesis regulation related BvMYB1 of beet, suggesting that they may play an important role in regulation of betalain biosynthesis in A. tricolor. Moreover, there were lots of genes were annoted as ’unknown genes’, indicating that there are still many unknown factors influencing betalain metabolism.